Test for hemolytic streptococci and test media therefor

ABSTRACT

The presence of beta hemolytic streptococci in a test culture is rapidly ascertained by inoculating a solid nutrient medium such as blood agar with the test culture, wherein the medium contains a ribonucleic acid preparation consisting essentially of ribonucleic acid core, and observing the medium for appearance of markedly enhanced hemolytic characteristics of the streptococci.

United States Patent 11 1 [111 3,781,192 Wood et a1. Dec. 25, 1973 [54]TEST FOR HEMOLYTIC STREPTDCOCCI 3,627,644 12/1971 Okamoto 81a]. 195/96AND TEST MEDIA THEREFOR 3,477,914 11/1969 Okamoto et al. 195/96inventors: Harrison F. Wood, 183 Livingston St., New Haven; SidneyBernstein, 49 Harbor View Ave., Milford, both of Conn.

Filed: May 1, 1972 Appl. No.: 248,816

US. Cl. 195/1011, 195/1035 R Int. Cl Cl2k 1/10 Field of Search 195/1035,99, 100,

References Cited UNITED STATES PATENTS 10/1972 Kronish et al 195/1035 RPrimary Examiner-A. Louis Monacell Assistant Examiner-Robert J. WardenAtt0rney-Anthony P. De Lio et al.

[5 7] ABSTRACT 8 Claims, No Drawings TEST FOR HEMOLYTIC STREPTOCOCCI ANDTEST MEDIA THEREFOR BACKGROUND OF THE INVENTION This invention relatesto a rapid and markedly enhanced test for the presence of hemolyticstreptococci and to test media therefor. More particularly, theinvention concerns a diagnostic test for beta hemolytic streptococciwhich will yield presumptive results usually within less than eighthours and which is adaptable for use by relatively unskilled personnelin hospitals, clinics, physicians offices and the like.

Lancefield Group A beta hemolytic streptococci are associated with awide variety of ailments in mammals, including such human ailments asrespiratory infections of various kinds. These bacteria are dangerouspathogens and the early determination of their presence is thereforeimportant for treatment of a patient afflicted with ailmentsattributable to these bacteria. For example, if their presence canquickly be determined, a medicinal specific to their control may beadministered before an illness reaches a serious level. Heretofore, apresumptive diagnostic test for these bacteria has required at least 18to 24 hours, thus permitting the proliferation of the bacteria while thediagnostic test is being completed, or promoting the interim use of medicinals which are not necessarily protective against the streptococcior which may have side effects which would have been avoidable if thediagnosis had been completed earlier.

The term hemolytic or like term means the rupture or lysis of mammalianred blood cells or corpuscles (erythrocytes) whereby the hemoglobin isreleased. This occurrence is observed in the form of transparent zones(lakes) at the original site of the cells. One of the three major groupsof Streptococcus bacteria, the so-called pyogenic (pus-forming) group,includes highly pathogenic bacteria which have the property of inducinghemolysis, and therefore their presence can be determined by observing asuspect culture for this occurrence. Under controlled conditions, thetwo subgroups of hemolytic streptococci can be distinguished: the S.pyogenes group and the S. viridans group, also known as beta hemolyticstreptococci and alpha hemolytic streptococci, respectively.

In the case of hemolysis induced by' beta-hemolytic streptococci, thezones of hemolysis appear around the streptococci colonies, and when thetest medium is bloor agar, the zones are entirely clear, almostcolorless (grey cast), and free from intact red blood cells. In the caseof alpha hemolytic streptococci, the nearly colorless zone of hemolysisis also observed, but in addition, there is a zone of discolored butintact red blood cells close in and around the streptococcal colonies.When blood agar is the test medium, the red blood cells in the region ofalpha hemolytic streptococci have a green or brownish green color. Whendeep colonies of streptococci in blood agar plates show no visiblechange in the blood cells surrounding the colony, the bacteria are saidto be of the gamma or non-hemolytic type.

Since it has been determined that the Group A beta hemolyticstreptococci are pathogens in the great majority of illnesses known toresult from the presence of streptococci in humans, it is apparent thata rapid diagnostic test for the presence of these bacteria will have farreaching benefits for improved health.

OBJECTS AND SUMMARY Accordingly, an object of the invention is toprovide a new and improved test for beta hemolytic streptococci havingthe characteristics of specificity of reaction, increased hemolyticreactions, simplicity (permitting use by non-specialized healthpersonnel), and rapid reading time.

Still another object is to provide a new and improved diagnostic testfor hemolytic streptococci having a reading time usually of 8 hours orless, and test media therefor.

Still another object is to provide a new and improved rapid diagnostictest for beta hemolytic streptococci, and test media therefor, which canbe used at a wide variety of locations including hospitals, clinics andphysicians offices.

These and other objects, features and advantages of the invention willbecome apparent from the specification which follows.

In summary outline, it has been discovered that the combination of asolid nutrient (growth) medium for hemolytic streptococci and aribonucleic acid preparation consisting essentially of ribonucleic acidcore material, markedly increases the rate of hemolysis when hemolyticstreptococci are present so that test results can be read at leastpresumptively usually within 8 hours. Since the preferred form of thetest medium of the invention (blood agar plate)is inexpensive and easilyobtainable, and since relatively little experience is necessary to readthe test results, the invention provides a procedure which can beeffectively utilized at health centers visited by the patients. As aconsequence, the usual procedure of taking a test culture from apatient, transporting it to another location for the diagnosis, and thenreporting the test results to the health officer and patient, can beabrogated with very substantial savings in time and expense. Theinvention therefore provides a significant contribution to thealleviation of illnesses associated with Group A beta hemolyticstreptococci.

The invention accordingly comprises the several steps and the relationof one or more of such steps with respect to each of the others, theapparatus embodying features of construction, combination andarrangement of parts which are adapted to effect such steps, and thearticle which possesses the characteristics, properties and relation ofelements, all as exemplified in the detailed disclosure hereinafter setforth, and the scope of the invention will be indicated in the claims.

DETAILED DESCRIPTION The solid nutrient medium is any medium whichprovides a growth environment for hemolytic streptococci and which willpermit the observation of typical hemolysis if it occurs. The mostextensive experience to date has been with agar formulated in known waysin admix ture with a nutrient material such as a beef broth infusion.Agar base media, particularly when used with Petri plates, aretransparent and colorless, are not digested or liquified by mostbacteria, melt only at boiling temperature, and once melted, do not setagain until body temperature has been reached. The agar generally ispresent in the nutrient material in amounts of about 1.5 to about 3.0percent by weight. Both natural and synthetic nutrient materials may beused, including meat (such as beef) or vegetable broths, whether of theextract or infusion types. Peptones are also common nutrients. Thenutrient medium may also be in dehydrated form and may contain syntheticnutrient substances such as certain carbohydrates, amino acids,phosphates and fementates.

In place of agar, solid surfaces may be used to provide the solidmedium. Among these may be mentioned highly porous membranes of certainplastic materials including cellulose acetate, colodian or the like. Inone such technique, a membrane is mounted on a perforated plate andsupported in a funnel-shaped vessel. The membrane then acts as a sieveto filter the microorganisms from a test fluid or from a dilute brothculture of the test fluid. After fluid has passed through the membrane,the organism is deposited on the surface and the membrane is removedfrom the vessel and placed on a disk or pad or blotting paper (which maybe saturated with a nutrient solution if the test solution did notcontain a nutrient). The nutrients then diffuse through the membrane andcause growth of colonies on the surface in the same manner as in agar.

The foregoing and other culturing techniques employing solid nutrientmedia are well known, such as described in Frobisher, Fundamentals ofMicrobiology, 8th Ed., W. B. Saunders Co. (1968), pages 38-44, and theinvention includes the use of such other media and the obvious variantsthereof.

To the nutrient medium is added defibrinated sheep red blood cells inthe amount generally of about 3 to percent by volume. While almost anytype of mammalian red blood cells may be used, such as defibrinatedhorse, rabbit or sheep blood cells, human blood cells normally shouldnot be used where the diagnosis requires distinction to be made betweenbeta and alpha hemolytic streptococci, because alpha hemolyticstreptococci, which produce alpha hemolysis on sheep blood, frequentlywill appear to be beta hemolytic on human blood plates, thus confusingthe diagnosis. Sheep red blood cells are preferred because they containan inhibitor for Hemophilus hemolyticus. This organism is oftendifficult to distinguish from beta hemolytic streptococci by grossinspection.

Also present in the solid nutrient medium is a yeast extract known asribonucleic core (hereinafter RNA core). This material is thelyophilized limit polynucleotide which remains after exhaustiveenzymatic hydrolysis of yeast ribonucleic acid (RNA) and removal ofdigestion products by dialysis. This material is commercially availableand its preparation is described by R. J. Hilmoe, J. Biol. Chem. 235,2117 (1960). RNA core is aply named since it is the limit nucleotiderather than a crude yeast nucleotide extract or a purified extract whichis available in varying degrees of purity.

In a standard mode of preparation of the solid nutrient medium of theinvention, a conventional blood agar base material (pre-prepared tocontain the required amount of beef nutrient infusion or suitablepeptones) is heated to boiling to dissolve the agar, and the solution isadjusted to pH 7.5 by the addition of a suitable buffering agent. Thesolution is then sterilized in an autoclave and cooled to about 50-60 C.RNA core is separately dissolved in a minimal amount of distilled waterand added to the cooled solution. The mixture is allowed to cool to45-50C. before adding 5 percent by volume defibrinated sheep red bloodcells. The mixture is then poured into sterile plates and allowed tosolidify and is then ready for inoculation with a test culture. Thethus-prepared blood agar plates may also be stored under refrigerationfor several weeks for use when and where desired.

The concentration of RNA core will depend somewhat upon the type ofsolid nutrient medium and the relative proportions of ingredients in themedium. For the standard blood agar plate, it has been determined thatabout 10-25 mg. percent (per cc. of total nutrient medium prior tosolidification) provides the best results. Amounts of RNA core in excessof this do not improve the readability, and it has been found thatamounts in the range of about 200 mg. percent actually inhibit thehemolysis.

The solid nutrient medium containing the RNA core and red blood cellsmay be inoculated with a test culture and observed for hemolysis usingany of the techniques well known in the art. For example, it isconventional to first streak the surface of the agar plate parallel to acrayon line which is marked on the bottom of the plate so as to dividethe plate in half. Sub-surface stabs with test inoculum are also made inthe medium, preferably along a line perpendicular to the crayon line.

The inoculation of the medium with the test culture can follow theaddition of the RNA core to the medium or the RNA core material may beadded after the inoculation with the test culture, for example as asolution of RNA core in distilled water, which solution is dropped intothe medium with an eye dropper, for example. Also, porous membranes suchas filter paper disks may be impregnated with the RNA core material andthese used in place of the RNA core solution.

Following inoculation with the test culture and RNA core material, theplates normally are inverted and incubated at 37C. If beta hemolyticstreptococci are present, hemolysis will be observed in the mediumusually within 8 hours and often much earlier in the area of thesub-surface stabs. The hemolysis will be clearly evident and littleexperience is required to discern it.

Many variations are possible on the foregoing techniques and aredescribed in the literature relating to solid nutrient medium culturingtechniques, for example, Frobisher, supra., pages 423-424; and P. F.Frank and M. L. Levinson, A Laboratory Manual of Bacteriological andSerological Procedures Used In The Identification of Beta Hemolyticstreptococci, Research Project MR 00509-13001, Bureau of Medicine andSurgery, Navy Department, Washington, D. C. (January, 1966 Revised),pages 7-14.

The mechanism whereby the hemolysis is markedly enhanced quantitativelyand increased in rate by the solid growth medium containing the RNA corematerial is not fully understood but it may be related in some manner tothe enhancement of production of the hemolytic toxin streptolysin S.This toxin has been implicated as the cause of hemolysis of red bloodcells and it is known that the presence of ribonucleic acid extracts ofyeast favor its production. However, studies of the relationship betweenstreptolysin S. production and yeast nucleic acid have been limited toliquid, nongrowth (resting cell) media containing crude yeast nucleicacid, as in Bernheimer, Formation of a Bacterial Toxin (streptolysin S)By Resting Cells, The Journal of Experimental Medicine, 90, No. 5, pages373-392 (1949), and to the use of RNA core as in US. Pat. Nos. 3,477,914and 3,627,644 to Okamoto et al.

Despite this knowledge, so far as is known, no studies have been made ofminimum times for hemolysis and the use of the reaction to ascertain thepresence of hemolytic streptococci in a test culture. Moreover, althoughcertain additives have been reported to en hance streptolysinS.production in non-growth liquid media (additives such as glucosaminehydrochloride, maltose, dextrose, cysteine hydrochloride, gibberellicacid, and thiamine hydrochloride), these additives have failed toenhance hemolytic activity in solid nutrient media. Therefore, itappears that the degree of hemolysis and the increased rate of hemolysisis not necessarily caused by increased streptolysin S.production, and itwas totally unexpected that the use of a solid growth medium, incombination with RNA core material, would give the significantimprovements observed in the media of the invention.

The following examples are intended as further illustration of theinvention but are not necessarily limiting thereof, except as set forthin the claims. All parts and percentages are by weight unless otherwiseindicated.

EXAMPLE 1 To a dehydrated blood agar base medium having a beef nutrientconfined therein, available from Baltimore Biological Laboratories asCode No. 01-127, was added an amount of a buffering solution sufficientto adjust the pH to 7.5. The resulting agar medium was dissolved andsterilized. After cooling to 50-60C., the RNA core was added as adistilled water solution thereof to provide between -25 mg. percent ofthe material in the still-liquid medium. The RNA core was obtained fromthe Worthington Biochemical Corporation, Catalog No. 3472. Then 5percent by volume of defibrinated sheep red blood cells was added to themedium which had cooled to 4042C. The thusprepared agar nutrient mediumas poured into Petri plates, 17-20 ml. per plate, and permitted tosolidify. The test plates (and identical plates but without the RNAcore, as controls) were streaked with swabs containing known betahemolytic cultures maintained in Stuarts Transport Medium. Sub-surfacestabs were also made in the inoculated medium with a sterile platinumloop, generally following the procedures described in Frank andLevinson, A Laboratory Manual of Bacteriological and SerologicalProcedures Used In The Identification Of Beta Hemolytic streptococci,supra,

Observations of six such sets of plates after 3% hours and after 6%hours incubation at 37C. are reported in Table 1 below, where stabindicates positive identification in a sub-surface area of the platesand the numeral indicates the relative widths of the hemolytic zones. Ineach instance, the control plates displayed no hemolysis before 12 hoursof incubation whereas most of those plates containing the RNA coreexhibited easily visible hemolysis in the subsurface areas after 3%hours, which hemolysis rapidly increased and was observable on thesurface after 6% hours.

TABLE I Specimen 3% hrs. 6% hrs. 1 Control Plate Test Medium 1+ stab 2+stab 2 Control Test 1 stab 2 3+ stab 3 Control Test stab l+ stab 4Control Test stab 1+ stab 5 Control Test stab 2+ stab 6 Control Test istab 2+ stab EXAMPLE 2 Control plates and test plates were preparedsubstantially as described in Example 1 but the RNA core material wasadded as a solution from an eye dropper to the agar medium after themedium had been inoculated with a known beta hemolytic streptococciculture. In this series of experiments, reported in Table II below,recipe A contained 50 mg. percent RNA core, recipe B contained mg.percent RNA core, and recipe C contained mg. percent RNA core. Thenutrient medium of each plate also contained 5 percent by volume sheepred blood cells and 0.2 g. percent maltose. The pH was adjusted to 7.5.Excellent hemolysis was observed after 6 hours with significantadditional enhancement of hemolysis upon further incubation, and wasessentially the same after 6 hours and 24 hours. The reference tosurface in Table II means a zone of hemolysis on the surface of theagar.

TABLE II Recipe 6 hours. 24 hours. A 3+ stab, 2+ surface 4+ stab, 4+surface despite overgrowth v of staphylococci B stab 2+ stab, multiplecolonies with surface hemolysis C l+ stab 4+ stab, 4+ surface EXAMPLE 3TABLE III 5% hrs. RNA Core 22 hrs. RNA Core Specimen Control 10 25 50Control 10 25 50 :t: :i: 1+ :l:

streptococci, characterized by marked enhancement of hemolysis andincreased rate of hemolysis, which comprises inoculating a solidnutrient medium with a culture suspected of containing saidstreptococci, wherein said medium contains red blood cells capable ofhemolyzing in the presence of said streptococci, said medium alsocontaining a ribonucleic acid preparation consisting essentially ofribonucleic acid core, and observing said medium for the appearance ofhemolytic characteristics of said streptococci.

2. A diagnostic test as in claim 1 wherein said solid nutrient mediumcomprises a mixture of agar and a meat extract or infusion.

3. A diagnostic test as in claim 1 wherein said red blood cells comprisedefibrinated sheep blood cells in an amount effective for inhibition ofgrowth of Hemophilus hemolyticus.

4. A diagnostic test as in claim 1 wherein said solid nutrient mediumcomprises a mixture of agar and a meat extract or infusion, said redblood cells are present in an amount of about 3-5 percent by volume ofsaid medium prior to solidification, and said ribonucleic acidpreparation is present in an amount of about 10-25 mg. percent per cc.of said medium prior to solidification.

5. A diagnostic test as in claim 2 wherein said nutrient medium alsocontains a peptone.

6. A diagnostic test medium for beta hemolytic streptococci whichcomprises a mixture of: (a) mammalian red blood cells capable ofhemolyzing in the presence of said streptococci; (b) a ribonucleic acidpreparation consisting essentially of ribonucleic acid core; and (c) asolid nutrient medium for said streptococci.

7. The test medium of claim 6 wherein said red blood cells aredefibrinated sheep red blood cells, and said solid nutrient medium is amixture of agar and a meat extract or infusion.

8. A test medium as in claim 7 wherein the nutrient medium also containsa peptone.

2. A diagnostic test as in claim 1 wherein said solid nutrient mediumcomprises a mixture of agar and a meat extract or infusion.
 3. Adiagnostic test as in claim 1 wherein said red blood cells comprisedefibrinated sheep blood cells in an amount effective for inhibition ofgrowth of Hemophilus hemolyticus.
 4. A diagnostic test as in claim 1wherein said solid nutrient medium comprises a mixture of agar and ameat extract or infusion, said red blood cells are present in an amountof about 3-5 percent by volume of said medium prior to solidification,and said ribonucleic acid preparation is present in an amount of about10-25 mg. percent per 100 cc. of said medium prior to solidification. 5.A diagnostic test as in claim 2 wherein said nutrient medium alsocontains a peptone.
 6. A diagnostic test medium for beta hemolyticstreptococci which comprises a mixture of: (a) mammalian red blood cellscapable of hemolyzing in the presence of said streptococci; (b) aribonucleic acid preparation consisting essentially of ribonucleic acidcore; and (c) a solid nutrient medium for said streptococci.
 7. The testmedium of claim 6 wherein said red blood cells are defibrinated sheepred blood cells, and said solid nutrient medium is a mixture of agar anda meat extract or infusion.
 8. A test medium as in claim 7 wherein thenutrient medium also contains a peptone.